- Email: [email protected]
Discovery, design and synthesis of the first reported potent and selective sphingosine-1-phosphate 4 (S1P4) receptor antagonists
Bioorganic & Medicinal Chemistry Letters 21 (2011) 3632–3636
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Discovery, design and synthesis of the first reported potent and selective sphingosine-1-phosphate 4 (S1P4) receptor antagonists Miguel Guerrero a, Mariangela Urbano a, Subash Velaparthi a, Jian Zhao a, Marie-Therese Schaeffer b,c, Steven Brown b,c, Hugh Rosen b,c, Edward Roberts a,c,⇑ a
Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States c The Scripps Research Institute Molecular Screening Center, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States b
a r t i c l e
i n f o
Article history: Received 15 March 2011 Revised 18 April 2011 Accepted 21 April 2011 Available online 28 April 2011 Keywords: S1P4 receptor antagonists S1P1–3,5 receptor family 5-Aryl furan-2-arylcarboxamide
a b s t r a c t Selective S1P4 receptor antagonists could be novel therapeutic agents for the treatment of influenza infection in addition to serving as a useful tool for understanding S1P4 receptor biological functions. 5-(2,5-Dichlorophenyl)-N-(2,6-dimethylphenyl)furan-2-carboxamide was identified from screening the Molecular Libraries-Small Molecule Repository (MLSMR) collection and selected as a promising S1P4 antagonist hit with moderate in vitro potency and high selectivity against the other family receptor subtypes (S1P1–3,5). Rational chemical modifications of the hit allowed the disclosure of the first reported highly selective S1P4 antagonists with low nanomolar activity and adequate physicochemical properties suitable for further lead-optimization studies. Ó 2011 Elsevier Ltd. All rights reserved.
Sphingosine-1-phosphate (S1P) is a pleiotropic lysophospholipid that is formed in various cells including platelets, mast cells and macrophages in response to various stimuli such as grow factors, cytokines and antigens. It serves as an important biological mediator that influences endothelial and lung epithelial integrity1,2 as well as lymphocyte recirculation.3–6 S1P exerts its biological effects via extracellular signaling through five G-protein coupled receptor subtypes, namely S1P1 through S1P5.7 While S1P1–3 receptors are expressed ubiquitously, S1P4 expression is highly restricted to hematopoietic and lymphatic tissue and S1P5 is expressed in the central nervous system.8 S1P4 is coupled to Gai and Gao proteins and activates the ERK, MAPK, PLC cascades.9 Local modulation of S1P receptors in the lungs has been shown to alter dendritic cell activation and accumulation in the mediastinal lymph nodes, resulting in blunted T cell responses and control of immunopathological features of influenza virus infection, without involvement of the S1P1 immunosuppressive activities.10 Reports showing that in dendritic cells S1P5 expression is very low whereas S1P4 is highly expressed,11 suggest that chemical activation of the S1P4 subtype in the airways may be effective at controlling the immunopathological response to viral infections. It has been reported that S1P4 is upregulated during megakaryocytes differentiation suggesting that S1P4 antagonists are a suitable target for reactive thrombocytosis.12 However, aspects of the biological role of S1P4 remain unclear partly due to the lack of ligands, neither
agonists nor antagonists, with high selectivity against the S1P1–3,5 subtypes. Herein we report on the discovery, synthesis and structure–activity relationships (SAR) of novel S1P4 selective antagonists as a valuable tool to elucidate the biological and pharmacological profile of the target receptor. In house high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository (MLSMR) collection identified a 5-aryl furan-2-arylcarboxamide derivative 1 (Fig. 1) as a S1P4 antagonist hit with acceptable in vitro potency/selectivity profile. With the aim to validate the identified hit, 1 was re-synthesized (Scheme 1) confirming IC50’s of 78 nM at S1P4, 14 lM at S1P5 and no activity at concentrations up to 25 lM towards S1P1–3 subtypes. However, inadequate values of logarithm of partition coefficient and total polar surface area (c Log P = 4.8, tPSA = 38.5) were calculated for 1. We sought to increase potency and reduce lipophilicity (c Log P and tPSA optimal values ranges of 2.5–3.5 and 60–90, respectively),13 through rational chemical modifications of
⇑ Corresponding author. Tel.: +1 858 784 7770; fax: +1 858 784 7745. E-mail address: [email protected] (E. Roberts). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.04.097
Figure 1.
3633
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636 Table 1 (continued) c Log Pb
tPSAb
IC50
H
4.5
38.3
6400
4l
H
6.2
38.3
253
4m
H
5.0
47.6
931
4n
H
4.0
47.6
589
4o
H
4.2
58.6
58
4p
H
4.9
47.6
80
4q
H
5.4
38.3
63
4r
Me
5.5
29.5
8100
4s
H
3.4
90.7
52
4t
H
3.8
58.6
21
4u
H
4.7
64.6
34
4v
H
3.8
64.3
25
4w
H
3.7
67.4
94
4x
H
4.7
41.6
348
4y
H
6.7
38.3
115
4z
H
4.0
67.8
44
Compd
R
4k
Ar
Scheme 1. Synthesis of 5-(2,5-dichlorophenyl)-2-arylcarboxamides 1, 4a–z. Reagents and conditions: (i) 2 (1 equiv), 3 (2 equiv), DIPEA (2 equiv), CH2Cl2, rt, 2–4 h, 60–95%.
the hit structure, thereby altering the predicted physicochemical properties and S1P4 binding affinity. 1 was formally fragmented into two regions to investigate its SAR (Fig. 1): (A) aryl ring C-linked to furan, (B) arylamide. The preparation of various analogs with modified substituents on the phenyl ring of region B was easily achieved as presented in Scheme 1. Coupling of commercially available acylchloride 2 with a variety of anilines provided, in good yields, a structurally and electronically diverse set of analogs 4a–4z. The obtained compounds were submitted to S1P4 functional assay (Table 1).14 Potency and lipophilicity were not significantly affected by attaching small alkylic groups on positions 2 and 6 (4b, 4c, 4f) Table 1 S1P4 antagonists (IC50, nM)a Compd
R
Ar
c Log Pb
tPSAb
IC50
4a
H
5.5
38.3
165
4b
H
5.2
38.3
41
4c
4d
4e
H
H
H
5.5
4.0
3.3
38.3
56.8
58.6
89
417
573
4f
H
6.2
38.3
105
4g
H
3.8
72.5
302
4h
H
5.1
38.3
169
4i
H
4.5
47.6
163 a Data are reported as mean for n = 3 determinations. NA = no active at concentrations up to 25 lM. b c Log P and tPSA values are obtained from ChemDraw 12.0 V.
4j
–CH2–CH2–
5.6
29.5
NA
compared to the hit. When polar substituents, hydrogen donors or acceptors, were attached in the same positions the potency decreased more substantially (4d, 4e, 4h, 4i). Similar activity to
3634
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636
Scheme 2. Synthesis of 5-aryl-N-(2,6-dialkylphenyl)furan-2-carboxamides 9a–9q. Reagents and conditions: (i) 5 (1 equiv), 6 (1.2 equiv), EDCl (1.2 equiv), HOBt (1.2 equiv), DMF, rt, overnight, 90%; (ii) 7 (1 equiv), 8 (1.5 equiv), Pd(PPh3)4 (0.1 equiv), 2 M aq NaCO3 (2 equiv), 1,4-dioxane, 80 °C, 4–6 h, 20–85%.
the hit was observed for the 2,4,6-trimethyl derivative 4q. Incorporating the amidic nitrogen into a five-membered ring suppressed completely the activity (4j). Two major reasons were formulated to explain this phenomenon: (a) the constriction of the C–N bond rotation, (b) the loss of hydrogen bond donor capability. To evaluate the contribution of the N–H hydrogen bond donor, a N-methylated derivative 4r was synthesized. 4r was approximately 128-fold less potent than the homolog 4q; therefore the hydrogen-bond donor capability of the amide group was hypothesized to be essential for the potency. Noteworthy, position 4 of the phenyl ring tolerated lipophilic substituents (4q, 4y) as well as polar and ionizable groups as observed for 4n, 4p, 4w, 4x, 4y, and 4o, 4s–4v, 4z, the latter showing enhancement of both potency and physicochemical properties. Particularly, the amine 4v(CYM50358) [c Log P = 3.8, tPSA = 64.4] and alcohols 4t [c Log P = 3.8, tPSA = 58.6], 4z [c Log P = 4.0, tPSA = 67.8] were more potent than the hit compound 1 with more desirable physicochemical properties. Next, SAR studies of the aryl ring A were performed while maintaining 2,6-dialkylanilines in the pendant moiety B. The general synthetic route to afford 9a–9q is outlined in Scheme 2. Amide coupling of 2-furoic acid 5 with appropriate aniline 6 followed by Suzuki cross coupling under standard conditions15 with a wide range of aryl boronic acids afforded 9a–9q. S1P4 activity of 9a–9q is reported in Table 2. The 2,5-dimethyl analogue 9i showed similar potency to the hit compound. Deletion of 2-chlorine (9b) had a higher impact on the loss of activity than the removal of 5-chlorine (9c) (6- vs 3-fold). Notably, the 2,4dichlorophenyl regioisomer 9a was 25- and 7-fold less potent than the hit and the mono-chlorinated 9c, respectively, thus indicating that substitution at position 4 was detrimental for the potency. 2,6-Dimethylated derivative 9j was found to be inactive probably due to the anti-coplanar orientation of the phenyl ring. In an attempt to reduce lipophilicity, polar substitutions at positions 2 and 5 were installed, but were found detrimental for the activity
Table 2 S1P4 antagonists (IC50, nM)a Compd
9a
R
H
Ar
c Log Pb
4.9
tPSAb
38.3
Table 2 (continued) c Log Pb
tPSAb
IC50
H
3.6
38.3
575
9e
H
4.0
38.3
308
9f
H
5.5
38.3
NA
9g
Me
2.4
78.8
NA
9h
Me
3.4
56.8
513
9i
H
4.3
38.3
64
9j
H
4.0
38.3
NA
9k
H
3.3
38.3
448
9l
H
3.5
38.3
286
9m
H
3.5
38.3
118
9n
H
4.3
38.3
515
9o
H
3.7
38.3
93
9p
Me
3.3
47.6
130
Compd
R
9d
Ar
IC50
2000
9b
H
4.4
38.3
497
9c
Me
4.4
38.3
269 a Data are reported as mean for n = 3 determinations. NA = no active at concentrations up to 25 lM. b c Log P and tPSA values are obtained from ChemDraw 12.0 V.
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636
3635
Scheme 3. Synthesis of molecules 15, 16. Reagents and conditions: (i) 10 (1 equiv), 11 (1.5 equiv), Pd(PPh3)4 (0.1 equiv), 2 M aq Na2CO3 (2 equiv), 1,4-dioxane, 80 °C, overnight; (ii) LiOH (1.6 equiv), THF/MeOH/H2O (2:2:1), rt, 3 h, 65% (over two steps); (iii) 12 (1 equiv), 13 or 14 (1.5 equiv), EDCl (1.5 equiv), HOBt (1.5 equiv), DMF, overnight, 60–70%.
(9e, 9f, 9g, 9h), suggesting that region A binds to a lipophilic pocket. Successively, a new set of analogs was prepared by replacing the phenyl ring. Interestingly, thiophene and furan rings were found to be good bioisosteres. The 3-thienyl 9k and 2-thienyl 9m analogs were slightly more potent than the phenyl derivative 9d. As the presence of either chlorine or methyl groups in positions 2 and 5 of the phenyl ring were found to be essential for the activity, methylated and chlorinated thienyl derivatives (9l, 9n and 9o) were synthesized. Interestingly, 4-methyl-3-thienyl 9l was 1.5-fold more potent than 9k (similar trend was observed in the phenyl series, 9c vs 9d) whereas 3-methyl-2-thienyl 9o was equipotent to 9m. Moreover, equipotency was found for 3-chlorophenyl 9b and 2-furanyl 9p compared to 5-chloro-2-thienyl 9n and thienyl 9m, respectively. Interestingly, thienyl and furanyl derivatives showed more suitable physicochemical properties (3.3 6 c Log P 6 4.3) within the hit class. To merge SAR studies of region A and B, hybrid molecules 15 and 16 (CYM50374) were synthesized (Scheme 3). 5-Bromofuran 10 underwent Suzuki cross coupling with thiophene boronic acid 11 followed by ester hydrolysis to afford carboxylic acid 12 in good yields. Amide coupling of 12 with the opportune anilines 13 and 14 yielded the final compounds in moderate yields. Indeed, 15 (c Log P = 3.0, tPSA = 58.6) and 16 (c Log P = 2.7, tPSA = 58.6) were potent S1P4 antagonists (IC50 = 46 and 34 nM, respectively), with lower lipophilicity compared to the hit compound. A set of the most active compounds was selected for functional assays at S1P1–3,5 subtypes (Table 3). Notably, all the selected compounds displayed an exquisite selectivity for the S1P4 receptor versus the other receptor subtypes; among them 4v (CYM50358) and 16 (CYM50374) showing the most suitable physicochemical prop-
Table 3 S1P selectivity counter screen (IC50, nM, percentage of inhibition)a,b,c
a
Compd
S1P4 IC50
S1P1 IC50
S1P2 IC50
S1P3 IC50
S1P5 IC50
4b 4c 4o 4p 4t 4v 4z 9o 15 16
41 89 58 80 36 25 44 93 46 34
NA NA 5300 20%c 45%c 6400 35%c 95%c 75%c 80%c
50%c NA 2800 2800 3000 3900 2400 2600 80%c 90%c
55%c NA 5400 30%c 60% 95% 70%c 20%c 10%c NA
50%c NA 3000 40%c 40% 5500 (90%)b 60%c 40%c NA NA
(90%)b (70%)b (85%)b (90%)b (90%)b
Data are reported as mean for n = 3 determinations. Percentage of inhibition. c Percentage of inhibition at 25 lM. NA = no active at concentrations up to 25 lM. b
erties were selected as lead compounds to initiate a lead-optimization program. In summary, we conducted a systematic SAR analysis of novel selective S1P4 antagonists based on 5-aryl furan-2-arylcarboxamide 1 scaffold, identified by our HTS efforts. Physicochemical properties (c Log P and tPSA) were calculated for a preliminary evaluation of drug-like properties. Notably, introduction of different ionizable and polar groups at position 4 of region B led to the identification of molecules of particular interest (4v and 16) with attractive in vitro biological profile and adequate physicochemical properties. As the first disclosure of S1P4 antagonists with low nanomolar potency and high selectivity against S1P1–3,5 receptor subtypes, the class of molecules herein reported represents a significant milestone that may allow experiments aimed to gain more insights into the biological functions of S1P4 in fundamental immunological processes. Details of more in-depth ongoing SAR for lead-optimization of the identified lead molecules will be communicated in due course. Acknowledgments This work was supported by the National Institute of Health Molecular Library Screen Center Network grant U54 MH084512A. We thank Mark Southern for data management with Pub Chem. References and notes 1. Marsolais, D.; Hahm, B.; Walsh, K. B.; Edelmann, K. H.; McGavern, D.; Hatta, Y.; Kawaoka, Y.; Rosen, H.; Oldstone, M. B. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1560. 2. Marsolais, D.; Rosen, H. Nat. Rev. Drug Discov. 2009, 8, 297–307. 3. Alfonso, C.; McHeyzer-Williams, M. G.; Rosen, H. Eur. J. Immunol. 2006, 36, 149. 4. Jo, E.; Sanna, M. G.; Gonzalez-Cabrera, P. J.; Thangada, S.; Tigyi, G.; Osborne, D. A.; Hla, T.; Parrill, A. L.; Rosen, H. Chem. Biol. 2005, 12, 703. 5. Sanna, G.; Liao, J.; Jo, E.; Alfonso, C.; Ahn, M.; Peterson, M. S.; Webb, B.; Lefebvre, S.; Chun, J.; Gray, N.; Rosen, H. J. Biol. Chem. 2004, 279, 13839. 6. Wei, S. H.; Rosen, H.; Matheu, M. P.; Sanna, M. G.; Wang, S.; Jo, E.; Wong, C.; Parker, I.; Cahalan, M. Nat. Immunol. 2005, 6, 1228. 7. Rosen, H.; Goetzl, E. J. Nat. Rev. Immunol. 2005, 5, 560. 8. Gräler, M. H.; Bernhardt, G.; Lipp, M. Genomics 1998, 53, 164; (b) Van Brocklyn, J. R.; Gräler, M. H.; Bernhardt, G.; Hobson, J. P.; Lipp, M.; Spiegel, S. Blood 2000, 53, 164; (c) Im, D. S.; Heise, C. E.; Ancellin, N.; O’Dowd, B. F.; Shei, G. J.; Heavens, R. P.; Rigby, M. R.; Hla, T.; Mandala, S.; McAllister, G.; George, S. R.; Lynch, K. R. J. Biol. Chem. 2000, 275, 14281. 9. Toman, R. E.; Spiegel, S. Neurochem. Res. 2002, 27, 619. 10. Marsolais, D.; Hahm, B.; Edelmann, K. H.; Walsh, K. B.; Guerrero, M.; Hatta, Y.; Kawaoka, Y.; Roberts, E.; Oldstone, M. B.; Rosen, H. Mol. Pharmacol. 2008, 74, 896. 11. Maeda, Y.; Matsuyuki, H.; Shimano, K.; Kataoka, H.; Sugahara, K.; Kenji Chiba, K. J. Immunol. 2007, 178, 3437. 12. Golfier, S.; Kondo, S.; Schulze, T.; Takeuchi, T.; Vassileva, G.; Achtman, A. H.; Gräler, M. H.; Abbondanzo, S. J.; Wiekowski, M.; Kremmer, E.; Endo, Y.; Lira, S. A.; Bacon, K. B.; Lipp, M. FASEB J. 2010, 24, 4701. 13. (a) Hughes, J. D.; Blagg, J.; Price, D. A.; Bailey, S.; DeCrescenzo, G. A.; Devraj, R. V.; Ellsworth, E.; Fobian, Y. M.; Gibbs, M. E.; Gilles, R. W.; Greene, N.; Huang, E.;
3636
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636
Krieger-Burke, T.; Loesel, J.; Wager, T.; Whiteley, L.; Zhang, Y. Bioorg. Med. Chem. Lett. 2008, 18, 4872; (b) Muchmore, S. W.; Edmunds, J. J.; Stewart, K. D.; Hajduk, P. J. J. Med. Chem. 2010, 53, 4830. 14. The activity was measured by using Tango™ EDG6-bla U2OS cells which contain the human Endothelial Differentiation Gene 6 linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a b-arrestin/
TEV protease fusion protein and a b-lactamase (BLA) reporter gene under the control of a UAS response element. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cellpermeable BLA substrate. 15. McAllister, L. A.; Hixon, M. S.; Kennedy, K. P.; Dickerson, T. J.; Janda, K. D. J. Am. Chem. Soc. 2006, 128, 4176.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Discovery, design and synthesis of the first reported potent and selective sphingosine-1-phosphate 4 (S1P4) receptor antagonists Miguel Guerrero a, Mariangela Urbano a, Subash Velaparthi a, Jian Zhao a, Marie-Therese Schaeffer b,c, Steven Brown b,c, Hugh Rosen b,c, Edward Roberts a,c,⇑ a
Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States c The Scripps Research Institute Molecular Screening Center, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States b
a r t i c l e
i n f o
Article history: Received 15 March 2011 Revised 18 April 2011 Accepted 21 April 2011 Available online 28 April 2011 Keywords: S1P4 receptor antagonists S1P1–3,5 receptor family 5-Aryl furan-2-arylcarboxamide
a b s t r a c t Selective S1P4 receptor antagonists could be novel therapeutic agents for the treatment of influenza infection in addition to serving as a useful tool for understanding S1P4 receptor biological functions. 5-(2,5-Dichlorophenyl)-N-(2,6-dimethylphenyl)furan-2-carboxamide was identified from screening the Molecular Libraries-Small Molecule Repository (MLSMR) collection and selected as a promising S1P4 antagonist hit with moderate in vitro potency and high selectivity against the other family receptor subtypes (S1P1–3,5). Rational chemical modifications of the hit allowed the disclosure of the first reported highly selective S1P4 antagonists with low nanomolar activity and adequate physicochemical properties suitable for further lead-optimization studies. Ó 2011 Elsevier Ltd. All rights reserved.
Sphingosine-1-phosphate (S1P) is a pleiotropic lysophospholipid that is formed in various cells including platelets, mast cells and macrophages in response to various stimuli such as grow factors, cytokines and antigens. It serves as an important biological mediator that influences endothelial and lung epithelial integrity1,2 as well as lymphocyte recirculation.3–6 S1P exerts its biological effects via extracellular signaling through five G-protein coupled receptor subtypes, namely S1P1 through S1P5.7 While S1P1–3 receptors are expressed ubiquitously, S1P4 expression is highly restricted to hematopoietic and lymphatic tissue and S1P5 is expressed in the central nervous system.8 S1P4 is coupled to Gai and Gao proteins and activates the ERK, MAPK, PLC cascades.9 Local modulation of S1P receptors in the lungs has been shown to alter dendritic cell activation and accumulation in the mediastinal lymph nodes, resulting in blunted T cell responses and control of immunopathological features of influenza virus infection, without involvement of the S1P1 immunosuppressive activities.10 Reports showing that in dendritic cells S1P5 expression is very low whereas S1P4 is highly expressed,11 suggest that chemical activation of the S1P4 subtype in the airways may be effective at controlling the immunopathological response to viral infections. It has been reported that S1P4 is upregulated during megakaryocytes differentiation suggesting that S1P4 antagonists are a suitable target for reactive thrombocytosis.12 However, aspects of the biological role of S1P4 remain unclear partly due to the lack of ligands, neither
agonists nor antagonists, with high selectivity against the S1P1–3,5 subtypes. Herein we report on the discovery, synthesis and structure–activity relationships (SAR) of novel S1P4 selective antagonists as a valuable tool to elucidate the biological and pharmacological profile of the target receptor. In house high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository (MLSMR) collection identified a 5-aryl furan-2-arylcarboxamide derivative 1 (Fig. 1) as a S1P4 antagonist hit with acceptable in vitro potency/selectivity profile. With the aim to validate the identified hit, 1 was re-synthesized (Scheme 1) confirming IC50’s of 78 nM at S1P4, 14 lM at S1P5 and no activity at concentrations up to 25 lM towards S1P1–3 subtypes. However, inadequate values of logarithm of partition coefficient and total polar surface area (c Log P = 4.8, tPSA = 38.5) were calculated for 1. We sought to increase potency and reduce lipophilicity (c Log P and tPSA optimal values ranges of 2.5–3.5 and 60–90, respectively),13 through rational chemical modifications of
⇑ Corresponding author. Tel.: +1 858 784 7770; fax: +1 858 784 7745. E-mail address: [email protected] (E. Roberts). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.04.097
Figure 1.
3633
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636 Table 1 (continued) c Log Pb
tPSAb
IC50
H
4.5
38.3
6400
4l
H
6.2
38.3
253
4m
H
5.0
47.6
931
4n
H
4.0
47.6
589
4o
H
4.2
58.6
58
4p
H
4.9
47.6
80
4q
H
5.4
38.3
63
4r
Me
5.5
29.5
8100
4s
H
3.4
90.7
52
4t
H
3.8
58.6
21
4u
H
4.7
64.6
34
4v
H
3.8
64.3
25
4w
H
3.7
67.4
94
4x
H
4.7
41.6
348
4y
H
6.7
38.3
115
4z
H
4.0
67.8
44
Compd
R
4k
Ar
Scheme 1. Synthesis of 5-(2,5-dichlorophenyl)-2-arylcarboxamides 1, 4a–z. Reagents and conditions: (i) 2 (1 equiv), 3 (2 equiv), DIPEA (2 equiv), CH2Cl2, rt, 2–4 h, 60–95%.
the hit structure, thereby altering the predicted physicochemical properties and S1P4 binding affinity. 1 was formally fragmented into two regions to investigate its SAR (Fig. 1): (A) aryl ring C-linked to furan, (B) arylamide. The preparation of various analogs with modified substituents on the phenyl ring of region B was easily achieved as presented in Scheme 1. Coupling of commercially available acylchloride 2 with a variety of anilines provided, in good yields, a structurally and electronically diverse set of analogs 4a–4z. The obtained compounds were submitted to S1P4 functional assay (Table 1).14 Potency and lipophilicity were not significantly affected by attaching small alkylic groups on positions 2 and 6 (4b, 4c, 4f) Table 1 S1P4 antagonists (IC50, nM)a Compd
R
Ar
c Log Pb
tPSAb
IC50
4a
H
5.5
38.3
165
4b
H
5.2
38.3
41
4c
4d
4e
H
H
H
5.5
4.0
3.3
38.3
56.8
58.6
89
417
573
4f
H
6.2
38.3
105
4g
H
3.8
72.5
302
4h
H
5.1
38.3
169
4i
H
4.5
47.6
163 a Data are reported as mean for n = 3 determinations. NA = no active at concentrations up to 25 lM. b c Log P and tPSA values are obtained from ChemDraw 12.0 V.
4j
–CH2–CH2–
5.6
29.5
NA
compared to the hit. When polar substituents, hydrogen donors or acceptors, were attached in the same positions the potency decreased more substantially (4d, 4e, 4h, 4i). Similar activity to
3634
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636
Scheme 2. Synthesis of 5-aryl-N-(2,6-dialkylphenyl)furan-2-carboxamides 9a–9q. Reagents and conditions: (i) 5 (1 equiv), 6 (1.2 equiv), EDCl (1.2 equiv), HOBt (1.2 equiv), DMF, rt, overnight, 90%; (ii) 7 (1 equiv), 8 (1.5 equiv), Pd(PPh3)4 (0.1 equiv), 2 M aq NaCO3 (2 equiv), 1,4-dioxane, 80 °C, 4–6 h, 20–85%.
the hit was observed for the 2,4,6-trimethyl derivative 4q. Incorporating the amidic nitrogen into a five-membered ring suppressed completely the activity (4j). Two major reasons were formulated to explain this phenomenon: (a) the constriction of the C–N bond rotation, (b) the loss of hydrogen bond donor capability. To evaluate the contribution of the N–H hydrogen bond donor, a N-methylated derivative 4r was synthesized. 4r was approximately 128-fold less potent than the homolog 4q; therefore the hydrogen-bond donor capability of the amide group was hypothesized to be essential for the potency. Noteworthy, position 4 of the phenyl ring tolerated lipophilic substituents (4q, 4y) as well as polar and ionizable groups as observed for 4n, 4p, 4w, 4x, 4y, and 4o, 4s–4v, 4z, the latter showing enhancement of both potency and physicochemical properties. Particularly, the amine 4v(CYM50358) [c Log P = 3.8, tPSA = 64.4] and alcohols 4t [c Log P = 3.8, tPSA = 58.6], 4z [c Log P = 4.0, tPSA = 67.8] were more potent than the hit compound 1 with more desirable physicochemical properties. Next, SAR studies of the aryl ring A were performed while maintaining 2,6-dialkylanilines in the pendant moiety B. The general synthetic route to afford 9a–9q is outlined in Scheme 2. Amide coupling of 2-furoic acid 5 with appropriate aniline 6 followed by Suzuki cross coupling under standard conditions15 with a wide range of aryl boronic acids afforded 9a–9q. S1P4 activity of 9a–9q is reported in Table 2. The 2,5-dimethyl analogue 9i showed similar potency to the hit compound. Deletion of 2-chlorine (9b) had a higher impact on the loss of activity than the removal of 5-chlorine (9c) (6- vs 3-fold). Notably, the 2,4dichlorophenyl regioisomer 9a was 25- and 7-fold less potent than the hit and the mono-chlorinated 9c, respectively, thus indicating that substitution at position 4 was detrimental for the potency. 2,6-Dimethylated derivative 9j was found to be inactive probably due to the anti-coplanar orientation of the phenyl ring. In an attempt to reduce lipophilicity, polar substitutions at positions 2 and 5 were installed, but were found detrimental for the activity
Table 2 S1P4 antagonists (IC50, nM)a Compd
9a
R
H
Ar
c Log Pb
4.9
tPSAb
38.3
Table 2 (continued) c Log Pb
tPSAb
IC50
H
3.6
38.3
575
9e
H
4.0
38.3
308
9f
H
5.5
38.3
NA
9g
Me
2.4
78.8
NA
9h
Me
3.4
56.8
513
9i
H
4.3
38.3
64
9j
H
4.0
38.3
NA
9k
H
3.3
38.3
448
9l
H
3.5
38.3
286
9m
H
3.5
38.3
118
9n
H
4.3
38.3
515
9o
H
3.7
38.3
93
9p
Me
3.3
47.6
130
Compd
R
9d
Ar
IC50
2000
9b
H
4.4
38.3
497
9c
Me
4.4
38.3
269 a Data are reported as mean for n = 3 determinations. NA = no active at concentrations up to 25 lM. b c Log P and tPSA values are obtained from ChemDraw 12.0 V.
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636
3635
Scheme 3. Synthesis of molecules 15, 16. Reagents and conditions: (i) 10 (1 equiv), 11 (1.5 equiv), Pd(PPh3)4 (0.1 equiv), 2 M aq Na2CO3 (2 equiv), 1,4-dioxane, 80 °C, overnight; (ii) LiOH (1.6 equiv), THF/MeOH/H2O (2:2:1), rt, 3 h, 65% (over two steps); (iii) 12 (1 equiv), 13 or 14 (1.5 equiv), EDCl (1.5 equiv), HOBt (1.5 equiv), DMF, overnight, 60–70%.
(9e, 9f, 9g, 9h), suggesting that region A binds to a lipophilic pocket. Successively, a new set of analogs was prepared by replacing the phenyl ring. Interestingly, thiophene and furan rings were found to be good bioisosteres. The 3-thienyl 9k and 2-thienyl 9m analogs were slightly more potent than the phenyl derivative 9d. As the presence of either chlorine or methyl groups in positions 2 and 5 of the phenyl ring were found to be essential for the activity, methylated and chlorinated thienyl derivatives (9l, 9n and 9o) were synthesized. Interestingly, 4-methyl-3-thienyl 9l was 1.5-fold more potent than 9k (similar trend was observed in the phenyl series, 9c vs 9d) whereas 3-methyl-2-thienyl 9o was equipotent to 9m. Moreover, equipotency was found for 3-chlorophenyl 9b and 2-furanyl 9p compared to 5-chloro-2-thienyl 9n and thienyl 9m, respectively. Interestingly, thienyl and furanyl derivatives showed more suitable physicochemical properties (3.3 6 c Log P 6 4.3) within the hit class. To merge SAR studies of region A and B, hybrid molecules 15 and 16 (CYM50374) were synthesized (Scheme 3). 5-Bromofuran 10 underwent Suzuki cross coupling with thiophene boronic acid 11 followed by ester hydrolysis to afford carboxylic acid 12 in good yields. Amide coupling of 12 with the opportune anilines 13 and 14 yielded the final compounds in moderate yields. Indeed, 15 (c Log P = 3.0, tPSA = 58.6) and 16 (c Log P = 2.7, tPSA = 58.6) were potent S1P4 antagonists (IC50 = 46 and 34 nM, respectively), with lower lipophilicity compared to the hit compound. A set of the most active compounds was selected for functional assays at S1P1–3,5 subtypes (Table 3). Notably, all the selected compounds displayed an exquisite selectivity for the S1P4 receptor versus the other receptor subtypes; among them 4v (CYM50358) and 16 (CYM50374) showing the most suitable physicochemical prop-
Table 3 S1P selectivity counter screen (IC50, nM, percentage of inhibition)a,b,c
a
Compd
S1P4 IC50
S1P1 IC50
S1P2 IC50
S1P3 IC50
S1P5 IC50
4b 4c 4o 4p 4t 4v 4z 9o 15 16
41 89 58 80 36 25 44 93 46 34
NA NA 5300 20%c 45%c 6400 35%c 95%c 75%c 80%c
50%c NA 2800 2800 3000 3900 2400 2600 80%c 90%c
55%c NA 5400 30%c 60% 95% 70%c 20%c 10%c NA
50%c NA 3000 40%c 40% 5500 (90%)b 60%c 40%c NA NA
(90%)b (70%)b (85%)b (90%)b (90%)b
Data are reported as mean for n = 3 determinations. Percentage of inhibition. c Percentage of inhibition at 25 lM. NA = no active at concentrations up to 25 lM. b
erties were selected as lead compounds to initiate a lead-optimization program. In summary, we conducted a systematic SAR analysis of novel selective S1P4 antagonists based on 5-aryl furan-2-arylcarboxamide 1 scaffold, identified by our HTS efforts. Physicochemical properties (c Log P and tPSA) were calculated for a preliminary evaluation of drug-like properties. Notably, introduction of different ionizable and polar groups at position 4 of region B led to the identification of molecules of particular interest (4v and 16) with attractive in vitro biological profile and adequate physicochemical properties. As the first disclosure of S1P4 antagonists with low nanomolar potency and high selectivity against S1P1–3,5 receptor subtypes, the class of molecules herein reported represents a significant milestone that may allow experiments aimed to gain more insights into the biological functions of S1P4 in fundamental immunological processes. Details of more in-depth ongoing SAR for lead-optimization of the identified lead molecules will be communicated in due course. Acknowledgments This work was supported by the National Institute of Health Molecular Library Screen Center Network grant U54 MH084512A. We thank Mark Southern for data management with Pub Chem. References and notes 1. Marsolais, D.; Hahm, B.; Walsh, K. B.; Edelmann, K. H.; McGavern, D.; Hatta, Y.; Kawaoka, Y.; Rosen, H.; Oldstone, M. B. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1560. 2. Marsolais, D.; Rosen, H. Nat. Rev. Drug Discov. 2009, 8, 297–307. 3. Alfonso, C.; McHeyzer-Williams, M. G.; Rosen, H. Eur. J. Immunol. 2006, 36, 149. 4. Jo, E.; Sanna, M. G.; Gonzalez-Cabrera, P. J.; Thangada, S.; Tigyi, G.; Osborne, D. A.; Hla, T.; Parrill, A. L.; Rosen, H. Chem. Biol. 2005, 12, 703. 5. Sanna, G.; Liao, J.; Jo, E.; Alfonso, C.; Ahn, M.; Peterson, M. S.; Webb, B.; Lefebvre, S.; Chun, J.; Gray, N.; Rosen, H. J. Biol. Chem. 2004, 279, 13839. 6. Wei, S. H.; Rosen, H.; Matheu, M. P.; Sanna, M. G.; Wang, S.; Jo, E.; Wong, C.; Parker, I.; Cahalan, M. Nat. Immunol. 2005, 6, 1228. 7. Rosen, H.; Goetzl, E. J. Nat. Rev. Immunol. 2005, 5, 560. 8. Gräler, M. H.; Bernhardt, G.; Lipp, M. Genomics 1998, 53, 164; (b) Van Brocklyn, J. R.; Gräler, M. H.; Bernhardt, G.; Hobson, J. P.; Lipp, M.; Spiegel, S. Blood 2000, 53, 164; (c) Im, D. S.; Heise, C. E.; Ancellin, N.; O’Dowd, B. F.; Shei, G. J.; Heavens, R. P.; Rigby, M. R.; Hla, T.; Mandala, S.; McAllister, G.; George, S. R.; Lynch, K. R. J. Biol. Chem. 2000, 275, 14281. 9. Toman, R. E.; Spiegel, S. Neurochem. Res. 2002, 27, 619. 10. Marsolais, D.; Hahm, B.; Edelmann, K. H.; Walsh, K. B.; Guerrero, M.; Hatta, Y.; Kawaoka, Y.; Roberts, E.; Oldstone, M. B.; Rosen, H. Mol. Pharmacol. 2008, 74, 896. 11. Maeda, Y.; Matsuyuki, H.; Shimano, K.; Kataoka, H.; Sugahara, K.; Kenji Chiba, K. J. Immunol. 2007, 178, 3437. 12. Golfier, S.; Kondo, S.; Schulze, T.; Takeuchi, T.; Vassileva, G.; Achtman, A. H.; Gräler, M. H.; Abbondanzo, S. J.; Wiekowski, M.; Kremmer, E.; Endo, Y.; Lira, S. A.; Bacon, K. B.; Lipp, M. FASEB J. 2010, 24, 4701. 13. (a) Hughes, J. D.; Blagg, J.; Price, D. A.; Bailey, S.; DeCrescenzo, G. A.; Devraj, R. V.; Ellsworth, E.; Fobian, Y. M.; Gibbs, M. E.; Gilles, R. W.; Greene, N.; Huang, E.;
3636
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636
Krieger-Burke, T.; Loesel, J.; Wager, T.; Whiteley, L.; Zhang, Y. Bioorg. Med. Chem. Lett. 2008, 18, 4872; (b) Muchmore, S. W.; Edmunds, J. J.; Stewart, K. D.; Hajduk, P. J. J. Med. Chem. 2010, 53, 4830. 14. The activity was measured by using Tango™ EDG6-bla U2OS cells which contain the human Endothelial Differentiation Gene 6 linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a b-arrestin/
TEV protease fusion protein and a b-lactamase (BLA) reporter gene under the control of a UAS response element. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cellpermeable BLA substrate. 15. McAllister, L. A.; Hixon, M. S.; Kennedy, K. P.; Dickerson, T. J.; Janda, K. D. J. Am. Chem. Soc. 2006, 128, 4176.